Analysis of Protein Complexes in Wheat Amyloplasts Reveals Functional Interactions among Starch Biosynthetic Enzymes      

Abstract

Protein-protein interactions among enzymes of amylopectin biosynthesis were investigated in developing wheat (Triticum aestivum) endosperm. Physical interactions between starch branching enzymes (SBEs) and starch synthases (SSs) were identified from endosperm amyloplasts during the active phase of starch deposition in the developing grain using immunoprecipitation and cross-linking strategies. Coimmunoprecipitation experiments using peptide-specific antibodies indicate that at least two distinct complexes exist containing SSI, SSIIa, and either of SBEIIa or SBEIIb. Chemical cross linking was used to identify protein complexes containing SBEs and SSs from amyloplast extracts. Separation of extracts by gel filtration chromatography demonstrated the presence of SBE and SS forms in protein complexes of around 260 kD and that SBEII forms may also exist as homodimers. Analysis of cross-linked 260-kD aggregation products from amyloplast lysates by mass spectrometry confirmed SSI, SSIIa, and SBEII forms as components of one or more protein complexes in amyloplasts. In vitro phosphorylation experiments with g-32 P-ATP indicated that SSII and both forms of SBEII are phosphorylated. Treatment of the partially purified 260-kD SS-SBE complexes with alkaline phosphatase caused dissociation of the assembly into the respective monomeric proteins, indicating that formation of SS-SBE complexes is phosphorylation dependent. The 260-kD SS-SBEII protein complexes are formed around 10 to 15 d after pollination and were shown to be catalytically active with respect to both SS and SBE activities. Prior to this developmental stage, SSI, SSII, and SBEII forms were detectable only in monomeric form. High molecular weight forms of SBEII demonstrated a higher affinity for in vitro glucan substrates than monomers. These results provide direct evidence for the existence of protein complexes involved in amylopectin biosynthesis.Starch is produced by the majority of higher plant species inside plastids and represents a major storage product of many of the seeds and storage organs produced agriculturally and used for human consumption as well as many important industrial applications. The starch granule is a complex polymeric structure with a hierarchical order, allowing efficient packing of large amounts of Glc into a water-insoluble form, and is composed of two distinct types of Glc polymer: amylose and amylopectin.Amylose comprises largely unbranched a-(1/4)-linked glucan chains and does not appear to participate in the formation of the ordered part of the matrix. Amylopectin is a branched glucan polymer typically comprising between 65% and 85% of the starch granule mass, produced by the formation of a-(1/6)-branch linkages between adjoining linear, i.e. a-(1/4)-linked, glucan chains. The polymodal distribution of glucan chain lengths and branch point clustering within amylopectin allows short-to intermediate-sized glucan chains to form double helices that can pack together in organized arrays, which are the basis of the semicrystalline natur...

A review of starch‐branching enzymes and their role in amylopectin biosynthesis

“…In cereals, the SBEII class is subdivided into two distinct gene products, SBEIIa and SBEIIb, each with different kinetic characteristics and tissue expression patterns. SBEII isoforms transfer shorter chains (DP [6][7][8][9][10][11][12][13][14] and prefer amylopectin as a substrate (29). These characteristics have also been shown in a variety of other plant species, for example, rice (Oryza sativa L.; ref.…”

“…However, recent evidence in cereal endosperm amyloplasts indicates that they can associate with other starch biosynthetic enzymes (7,8) and can also form dimers (see "Regulation of SBE Activity" section; ref. 9.…”

“…Furthermore, in vitro dephosphorylation reduced the activity of SBEIIa and SBEIIb in amyloplasts and SBEIIa in chloroplasts, but had no measurable effect on the activity of SBEI (7). Studies with wheat and maize amyloplasts have shown that all SBE isoforms can be phosphorylated (8,9) and that protein phosphorylation regulates the association of SBE and SS isoforms in multienzyme protein complexes (see below; refs. 9,68,69).…”

“…An emerging aspect of our understanding of the regulation of storage starch biosynthesis is the physical association of different classes of amylopectin-synthesizing enzymes in protein complexes within starch-synthesizing plastids and the important regulatory role played by protein phosphorylation (7,9). The SBEII class forms a functional trimeric protein complex with SSI and SSIIa, and this complex is implicated in Phosphorylation sites on maize SBEIIb and SBEIIa.…”

Comparative proteome analysis of A- and B-type starch granule-associated proteins in bread wheat (Triticum aestivum L.) and Aegilops crassa

“…Protein-protein interaction among SS and BE isozymes in endosperm form wheat [40,71] and maize [72] was reported recently. These strongly suggest that each enzyme involved in starch biosynthesis operates as part of the functional multi-protein complexes; some enzymes' functions could be replaced by their isoforms and widely occurred protein-protein interactions.…”